The sponge used for this experiment was the demospongiae Amphimedon sp. of the family niphatidae (fig 1). The individuals were sampled off the East Coast of Australia, and put in an aquaria. A small amount of tissue was removed from two sponges and checked under a compound microscope, to see if the individuals were alive before the experiment started. Nine live individuals of similar volume (≈1000mm³) were picked out. The volume was measured using a caliper (±0.01mm). For the study four different particles were filtrated by the sponges: Microplastics of the size 10µm and algae of the sizes Nano (<2µm), 4-7µm and 7-20µm. The experiments took place during two different lab sessions two weeks apart. The first week no plastics were filtrated. The sponges were separated into individual cups with 100ml of seawater (this was enough to cover all the sponges in water). They were then separated into three different groups, i.e. 3×3 sponges. The first group were exposed to algae of the Nano size, the second group 4-7µm sized algae and the third group 7-20µm.  2 µl of each algal concentration was added to the cups (fig 2). The algae was stirred, then 1ml of the solution was removed and put into cuvettes, for absorbance testing. This was the 0 hour mark. Three cuvettes were used per cup, to account for the variance of the spectrophotometer. This procedure was repeated at the 1 hour and 2 hour mark. The algae was kept suspended in the liquid by stirring the cup every 10 min. The clearance rate was measured using a spectrophotometer, using OD 600nm. Once the data was recorded, the contents of the cuvette were poured back into the cup.

After two weeks a similar experiment was carried out. Here the Nano sized algae was replaced with microplastics of the size 10µm. This size was chosen due to the results of the first experiment. The absorbance was tested every half hour in this experiment, which ran for two hours. Again, there was in total 3×3 sponges. One group was fed microplastics 10µm, one was fed 4-7µm algae and the third group was fed 7-20µm algae. The cups were stirred every 10min. For the spectrophotometer 1ml of the solution was put in a cuvette, three cuvettes per cup was used, to account for the variance of the spectrophotometer. Before the absorbance was read, the cuvettes were manually shaken in order to suspend any particles that had settled at the bottom of the cuvette. Once the data was recorded, the contents of the cuvette were poured back into the cup.

For the experiment, 2µl of highly concentrated algae was added to 100ml of sea water. In order to find roughly how many algal cells were in each cup, a hemocytometer was used. The hemocytometer was loaded with 10µl of the particle mixed sea water. The hemocytometer was then placed on a compound microscope, where the number of cells were counted, using the hemocytometer’s grid lines. From this, the number of cells per 1ml of sea water could be calculated. Plastic: 33000cells/ml, 4-7µm: 22000cells/ml, 7-20µm: 30000cells/ml.

In order to identify the sponge, the spicules had to be extracted. This was done by covering a small piece of the sponge with bleach, in an Eppendorf tube. The solution was left for approximately 40min, until the tissue was dissolved. The solution was then extracted and put on a slide, and checked using a Compound microscope.

In order to tell what happened with the plastics in the aforementioned experiment, one sponge was pressed through a filter, in order to try and isolate the choanocyte chambers. The sponge contents were mixed in salt water, the solution was then put on a slide and checked using a compound microscope. Photographs were taken, as a record of the results.

The data was analyzed using Microsoft Excel 2013, where the data was plotted in scatterplot graphs. Exponential trendlines were calculated along with r² values.

This study have found that there generally is a very small difference in the clearance rate between different sized algae, using the same sized sponges (fig 4). However the trend that could be noticed, was that the medium sized cells 4-7µm were cleared faster than the other algal sizes, with the Nano sized cells (<2µm) being filtered the slowest. The results could suggest that food particles are taken up slower by the choanocytes compared to the pinacocytes lining the channel walls of the sponge. Previous studies have shown that the particle sizes less than 0.2 microns are generally filtered out slower compared to larger micron sized particles (Turon, 1997). Turon (1997) found, however, that particles of the size 1µm was retained the most efficient, which disagrees with the results in this study. However, it is possible that the Nano sized algae contained a large percentage of cells smaller than 1µm. This should be investigated for future research. Worth noting, is that the sponges used in the Turon (1997) experiment were different from the species used here. Now, the data in this study was very limited in terms of replicates, and so to further investigate the relationship between particle size and clearance rate, I suggest having more replicates. This will grant more statistical power. Interestingly, if the theory of different sized particles being picked up at different locations in the sponge, either pinacocysts or choanocytes, is applicable for the Amphimedon sp. then the results (fig 4) suggests that the efficiency of retaining particles is not very different between the two systems. For further research I suggest looking at the pinacocysts and choanocytes to see if different sized particles are found at these locations.

The study found that regardless of the size of the algae, the clearance rate of the sponges followed an exponential curve (fig 4, 5, 6). This was expected, since the sponges are filter feeders and not actively seeking the food particles. So when the algal concentration is high in the beginning, a higher percentage of the food particles will be sucked in by the flagellar movement of the choanocyte chamber. As the concentration of algae decreases, less and less algae will be close enough to the sponge to be caught by the ingoing current. This agrees with (Turon, 1997). If the contents of the cup would have been continuously stirred, then it could have potentially impacted the slope of the clearance rate. Since the sponge would be more regularly exposed to the algae. In order to get a better picture of how many cells was retained by the sponges, I suggest counting the actual number of cells in the cups. This is most likely a more reliable way of measuring the clearance rate. Another way of measuring the amount of cells could be to measure the dry weight of the sponge, however this implies that the algal cells way the same, and that the sponges are of equal size.

This study also found that microplastics are soaked up by the sponge (fig 7 & 8). An attempt at locating intact choanocyte chambers was made, unfortunately none were found. Thus the only conclusion that can be draw from figure 5, 6 and 7 is that the plastics seem to be taken up by the sponge. However, where exactly they go in the sponge and how it affects the sponge is still unknown. For further research I suggest to try and find intact choanocyte chambers, and see if the plastics are able to enter the choanocytes, or potentially clog them There is evidence that a high concentration of food particles can clog the pores of the sponge (Maldonado et al., 2010). Whether or not microplastics enables clogging or not needs to be researched. Another follow up experiment would be to test how plastic exposed sponges compare in clearance rate against control sponges. This would enable us to further understand how microplastics may affect the clearance rate of sponges. Furthermore, allowing the plastic covered sponge to recover in a healthy environment, could tell us more about the sponges ability to rid itself of plastics. It is important to note that the microplastic concentration used in this experiment was significantly higher than what we would expect to find in the ocean. However the results do show that the plastics can take up microplastics, and to some extent figure 5 and 6 show that the sponges can exclude some amount of the plastics, once they’ve entered the sponge. However, the results suggest that far from all plastic particles are excluded by the sponge.  This could potentially lead to a scenario where sponges have to spend more and more energy, as the microplastic concentration rises, to get rid of the unwanted particles. This could result in less food uptake by the sponge.

The choice of measuring the optical density for 600nm was made as we would expect the turbidity of the solution to decrease as algae and plastics were consumed by the sponge. However, the light will scatter differently depending of the size of the cell. Furthermore, for the second experiment, although the same amount of algae was used as in the first experiment (2µl), the spectrophotometer recorded very different absorbances for the 0h mark. This was very surprising, and could indicate a malfunction of the spectrophotometer, or some sort of human error. In order to get more reliable data I suggest counting the number of particles in the 100ml cup over time, and set up the experiment to have the same amount of particles in each replicate.

Another issue that was encountered during the experiment was that the microplastics sank very fast once they were added to the seawater. This may have had two implications. Firstly, it is possible, that the plastic beads spent most of the time at the bottom of the cup, not being able to be soaked up by the sponge. Continuous stirring, or shortening the interval between stirs, instead of the 10min interval that was used this study, may have allowed the sponges to have a better chance of filtrating the microplastics. Secondly, the rapid sinking rates may explain the generally larger standard deviation found in the microplastic data (fig 5 & 6). As the water was added to the cuvette, the plastics might have sunk down to the bottom of the cuvette, before the absorbance could be measured. This was, however, accounted for by manually shaking the cuvettes before the OD 600nm was measured. For future research automatically stirring of the cups, using a magnetic stirrer for instance, along with more replicates and a rapid read of the OD 600nm should be implemented.

This study found that there is a very small difference in clearance rate between particle sizes Nano (<2µm), 4-7µm and 7-20µm for the sponge Amphimedon sp. the Different particle sizes are normally picked up by the sponge on different locations, pinacocytes and choanocytes. This study concludes that there is a very small difference in retention rates between these two sites, however that the Nano (<2µm) are retained the slowest, which agrees with previous studies (Turon, 1997), and that the 4-7µm sized algae are picked up the fastest. The study also found that microplastics of the size 10µm do not behave exactly the same way as algal cells of similar size. The data suggests that the sponges can recognize plastic as a nonfood particle and, to some extent, dispose of it. Sponges being highly specific in their food uptake have been shown before (Wehrl et al., 2007). The results showed that the majority of the microplastics were taken up by the sponge, however. Further research is needed on what effect this will have on sponges in the long term.